Our dream of watching a protein function in real time with 150 ps time resolution and near-atomic spatial resolution was first realized in 2003 using picosecond time-resolved Laue crystallography, an experimental methodology developed by the Anfinrud group at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. To advance this capability further, we initiated in 2005 a major effort to develop the infrastructure required to pursue picosecond time-resolved X-ray science on the BioCARS 14-IDB beamline at the Advanced Photon Source (APS) in Argonne, IL. That effort, which has been highly successful, is documented in a separate report. We acquire time-resolved Laue diffraction images using the pump-probe method. Briefly, a laser pulse (pump) photoactivates a protein crystal, after which a suitably delayed X-ray pulse (probe) passes through the crystal and records its diffraction pattern on a 2D detector. Because we use a polychromatic X-ray pulse, we capture thousands of reflections in a single image without having to rotate the crystal. This Laue approach to crystallography boosts substantially the rate at which time-resolved diffraction data can be acquired. The information needed to determine the proteins structure is encoded in the relative intensities of the diffraction spots observed. However, the structural information contained in a single diffraction image is incomplete, requiring repeated measurements at multiple crystal orientations to produce a complete set of data. We continue to develop TReX, an in-house software package designed to analyze time-resolved Laue data, and are working on TReX-II, a major update to this software package that employs a ratio method for data processing. The ratio method is based upon back-to-back diffraction images acquired with (ON) and without (OFF) a laser pump pulse. By merging ION/IOFF ratios of integrated spot intensities for each indexed reflection, instead of intensity differences, errors arising from image scaling and wavelength normalization are avoided. After merging, the ratios are easily converted to structure factor amplitude differences, which are needed for structure refinement. We have used photoactive yellow protein (PYP) as a model system for understanding how signaling proteins function. PYP is a 14-kD water-soluble blue-light receptor that, upon absorbing a single photon, produces a signaling state that causes a purple sulfur bacterium, Halorhodospira halophile, to swim away from blue light. The chromophore in PYP is p-coumaric acid (pCA), which is covalently linked to the Cys69 residue via a thioester bond. Its C2=C3 double bond is trans in the ground state, but upon absorbing a photon, is converted to cis with modest quantum efficiency. This photoisomerization event triggers a sequence of structural changes that involve spectroscopically red-shifted (pR) and blue-shifted (pB) intermediates, the last of which corresponds to the putative signaling state. We have used time-resolved Laue crystallography to investigate the structures of the intermediates produced as this protein progresses through its fully reversible photocycle, and published these results in the Nov 12, 2012 issue of PNAS. This effort proved quite challenging. The PYP crystals proved to be radiation sensitive, which limited the number of diffraction images that could be acquired from a single protein crystal. To mitigate the adverse effects of radiation damage, we spread the X-ray/laser dose over the entire length of large crystals, and acquired complete time series at several different orientations with each crystal. With numerous crystals, we achieved sufficient completeness to generate high-resolution structures over a complete time series spanning 10 decades of time with 150 ps time resolution. This study unveiled a simple, stepwise structural progression of PYP conformations toward a long-lived pB0 state, with each transition characterized at an unprecedented level of detail. The highly contorted pR0 state provides a visual clue regarding the conformational gymnastics that must accompany trans/cis isomerization in a highly constrained protein environment. The novel pR0 structure unveiled through this work corresponds to a highly strained cis intermediate that launches the PYP photocycle. The pCA carbonyl in pR0 is oriented 90 out of the plane of the phenolate and appears to be locked in this twisted conformation by the hydrogen bond between this carbonyl and the Cys69 backbone nitrogen. In a collaboration with Dr. Gerhard Hummer, this unusual conformation was examined by Density Functional Theory (DFT) calculations and found to be chemically plausible. Moreover, the X-ray refined and DFT-optimized structures were found to be in excellent agreement for all other intermediates as well, thereby cross-validating these structures and confirming the plausibility of our pR0 structure. The structure of our pR0 intermediate was later contradicted by another study published in Nature Chemistry in 2013. In collaboration with Dr. Gerhard Hummer, we published in the March 2014 issue of Nature Chemistry a comment that addressed this contradiction, and presented new results that not only support our pR0 structure, but also illuminated flaws in the analysis that led to the contradictory structure. For details, see Contradictions in X-ray structures of intermediates in the photocycle of photoactive yellow protein, Ville R. I. Kaila, Friedrich Schotte, Hyun Sun Cho, Gerhard Hummer & Philip A. Anfinrud, Nature Chemistry, 6, 258 (2014). We also published a time-resolved Laue study of hemoglobin, which allowed us to track the time-dependent population of toxic CO in the primary docking sites following laser photolysis. For details, see Real-time tracking of CO migration and binding in the &#945; and &#946; subunits of human hemoglobin via 150-ps time-resolved Laue crystallography, Friedrich Schotte, Hyun Sun Cho, Jayashree Soman, Michael Wulff, John S. Olson and Philip A. Anfinrud, Chemical Physics, 422, 98-106 (2013). In November 2013, we attempted a femtosecond time-resolved X-ray diffraction study of a heme protein at the LCLS in Stanford, California. The LCLS is the world's first X-ray free electron laser, and produces intense X-ray pulses shorter than 100 fs in duration (<10-15 s). For this study, we designed a novel high-speed X-ray diffractometer and installed it on the XPP beam line at the LCLS. This study proved quite challenging, as we attempted to use a high-speed X-ray detector for the first time, and had to develop beamline control software capable of synchronizing our diffractometer with the X-ray laser used to probe the protein crystal, and the optical laser used to photo excite the protein crystal. Moreover, we had to develop a protocol for scanning the protein crystal through the X-ray beam to find its edge to a precision of at least 10 m. Though we succeeded in getting the infrastructure functioning properly, there wasn't sufficient beam time left to acquire the amount of data needed to produce a femtosecond time-resolved movie of the structural changes occurring in the protein crystal. We have submitted a proposal for another week of LCLS beam time to finish this experiment. By capturing the structure and temporal evolution of key reaction intermediates, time-resolved Laue crystallography provides an unprecedented view into the relations between protein structure, dynamics, and function. Such detailed information is crucial to properly assess the validity of theoretical and computational approaches in biophysics, and allows us to unveil reaction pathways that are at the heart of biological functions.